Process for the preparation of cilastatin

ABSTRACT

The present invention relates to an efficient and industrially advantageous process for the preparation of pure cilastatin.

FIELD OF THE INVENTION

The present invention relates to an efficient and industriallyadvantageous process for the preparation of pure cilastatin.

BACKGROUND OF THE INVENTION

Cilastatin possesses the ability to prevent nephrotoxicity associatedwith the use of β-lactam antibiotics such as imipenem. Chemically,cilastatin is [R═{R*,S-(Z)]]-7-[(2-amino-2-carboxyethyl)thio]-2-[[2,2-dimethylcyclopropyl)carbonyl]amino-2-heptenoicacid and has the structural Formula I.

It is a renal dehydropeptidase inhibitor and is co-administered as thesodium salt with imipenem in order to prevent its renal metabolism.Imipenem/cilastatin combination is used as a potent broad spectrumantibacterial agent.

Cilastatin was first disclosed in U.S. Pat. No. 5,147,868 and wasobtained in a multi-step synthesis involving condensing cysteinehydrochloride with heptenoic acid of Formula II,

wherein X is chloro or bromo, in the presence of sodium hydroxide inaqueous medium. Cilastatin so obtained contains the correspondingundesired E-isomer in amounts ranging from about 6 to 10% as determinedby HPLC. A process for isomerising the E-isomer to cilastatin by heatingthe mixture at pH 3 is also disclosed. However, we have observed thatthe isomerization process results in the formation of impurities in therange of 5-8% due to the degradation of cilastatin which renders theproduct produced by this process unsuitable for human consumption.

U.S. Pat. No. 5,147,868 also teaches a method for isolating cilastatinfrom the reaction mixture involving two purifications viz.chromatography using a cation exchange resin followed by solventpurification using ethanol and diethyl ether. The ion exchangechromatography removes inorganic salts such as sodium chloride which isotherwise difficult to remove as cilastatin itself is also watersoluble.

However, our attempts to isolate pure cilastatin by following theprocess exemplified in said patent were unsuccessful. We could obtaincilastatin in the form of its ammonium salt on eluting the cationexchange resin with ammonia solution and not as the free acid. Obtainingthe free acid by using an acid such as hydrochloric acid entailedformation of inorganic ammonium salts such as ammonium chloride thusdefeating the very purpose of using a cation exchange resin.

J. Med. Chem. 1987; 30: 1083 discloses a process for the preparation ofcilastatin involving the condensation of cysteine with thehalo-heptenoic acid of Formula II in sodium metal/liquid ammonia and theresultant mixture is isomerized to obtain cilastatin using methyl iodidein methanol. Cilastatin is isolated by using a cation exchange resinfollowed by the treatment with an anion exchange resin to remove theinorganic salts.

However, this process is not suitable on an industrial scale as itinvolves the use of sodium metal/liquid ammonia which are very hazardousand also uses methyl iodide for isomerization, which is expensive andrequires special storage conditions. More over, loading the ion exchangecolumn requires repeated circulation of cilastatin solution. Also, ionexchange column operates on the principle of ionic bonding/acid basereaction. Such reaction being exothermic causes considerable degradationof cilastatin. The use of two stage ion exchange chromatography iscumbersome, tedious and not practicable on an industrial scale.

In light of the above drawbacks in the prior art processes, there is aneed for the development of a simple, convenient and efficient processfor the preparation of pure cilastatin which is convenient to operate onan industrial scale.

SUMMARY OF THE INVENTION

The present invention provides a process for the purification ofcilastatin using a non-ionic adsorbent resin. The process requires asingle purification using chromatographic technique to obtain the pureproduct. Loading of the non-ionic adsorbent resin with crude cilastatinis achieved by passing the solution only once through the resin. Sinceno acid base reaction takes place, no degradation of the product isobserved. The present invention thus fulfills the need for a processwhich is convenient to operate on an industrial scale.

Accordingly, the present invention provides a process for thepurification of cilastatin which comprises contacting a solution ofcrude cilastatin with a non-ionic adsorbent resin and recovering purecilastatin from a solution thereof.

In the meaning of the present invention, the term “crude cilastatin”comprises cilastatin containing impurities which may be inorganic saltssuch as sodium chloride, sodium bromide and the like, or organicimpurities which may have formed due to the degradation of cilastatin,or the side products formed during the synthesis, or unreactedintermediates of the multi-step synthesis for the preparation ofcilastatin.

The solution of crude cilastatin may be obtained by dissolving the crudecilastatin in a suitable solvent or may be obtained directly from thereaction mixture for the preparation of cilastatin containing alreadydissolved crude cilastatin. The term “suitable solvents” as used hereinincludes water, organic solvents, and mixtures thereof. The organicsolvents include methanol, ethanol, acetonitrile, acetone, and the like.The crude cilastain may be prepared by any of the methods reported inprior art.

Any of the non-ionic adsorbent resins which are commercially availableand on the surface of which cilastatin is adsorbed, may be used. Inparticular, non-ionic macroporous water insoluble polymers such aspolyacrylates or copolymers of styrene and polyvinyl benzene may beused. Preferred adsorbent resin is a copolymer of styrene cross linkedwith divinylbenzene.

In the meaning of the present invention, the term “pure cilastatin”refers to cilastatin having a purity of 98% or more by HPLC.

A typical process for the purification of cilastatin comprises loading asolution of crude cilastatin on a column of non-ionic adsorbent resin,washing it with deionized water till no halide ions can be detected. Theresin is then eluted with organic or aqueous organic solvent and purecilastatin is isolated from the eluate by common methods known in theart such as concentration, precipitation and recrystallization asrequired. However, alternative method of purification such as slurryingwith the adsorbent resin may also be used.

According to another aspect of the present invention, it provides aprocess for the isomerization of E-isomer to cilastatin. The processcomprises heating a solution of cilastatin containing the correspondingundesired E-isomer at a pH of about 0.5 to 1.5. We have observed thatcilastatin obtained using this process greatly reduces the formation ofdegradation products.

The solution of cilastatin containing the corresponding E-isomer ispreferably obtained directly from the reaction mixture for thepreparation of cilastatin. Cilastatin may be prepared by any of themulti-step processes described in prior art.

The isomerization is preferably performed at 85-95° C. The pH isadjusted to 0.5 to 1.5, more preferably to 0.5 to 1 and most preferablyto about 0.5. Any acid may be used for adjusting the pH of the solution.Preferably, hydrochloric acid is used.

In a preferred embodiment of the present invention, the two aspects ofthe invention are combined i.e. the isomerization process is followed bythe purification process to obtain pure cilastatin.

DETAILED DESCRIPTION OF THE INVENTION

In the following section preferred embodiments are described by way ofexamples to illustrate the process of the invention. However, these arenot intended in any way to limit the scope of the present invention.

EXAMPLE 1

Preparation of Cilastatin

Cysteine hydrochloride monohydrate (166.3 g) was dissolved in water (1.2L). To this solution, aqueous sodium hydroxide (113.7 g in 400 ml water)and sodium salt of7-chloro-2-[[(1S)-2,2-dimethylcyclopropane]carboxamido]-2-heptenoic acid(200 g) were added. The reaction mixture was stirred at roomtemperature. The corresponding E isomer (5% by HPLC) was isomerized tocilastatin by heating the reaction mixture at 85-90° C. for 30 minutesafter adjusting the pH to 0.5 with concentrated hydrochloric acid.

Purification of Cilastatin

The reaction mixture obtained above was loaded on a column packed withdiaion HP-20 resin as the adsorbent. The column was washed with water toremove sodium chloride and then the product was eluted with aqueousmethanol. The column fractions containing the pure product were pooledand concentrated to obtain pure cilastatin (160 g; Purity: 99% by HPLC).

EXAMPLE 2

Ethyl-7-chloro-2-oxo-heptanoate (25 g), (S)-2,2-dimethylcyclopropanecarboxamide (13.68 g) and p-toluene sulphonic acid (0.125 g) wererefluxed in toluene using dean—stark trap for the azeotropic removal ofwater from the reaction mixture. After the condensation was complete,the reaction mixture was washed with dilute hydrochloric acid andaqueous sodium bisulfite to remove the unreacted(S)-2,2-dimethylcyclopropane carboxamide andethyl-7-chloro-2-oxo-heptanoate, respectively. The organic solution wasthen concentrated to recover toluene under reduced pressure. Theresultant oily ethyl ester of7-chloro-2-[[(1S)-2,2-dimethylcyclopropane]carboxamide]-2-heptenoic acidwas hydrolyzed with aqueous sodium hydroxide in the presence ofdenatured spirit at room temperature. After hydrolysis, the reactionmixture was concentrated to half of its volume under reduced pressureand washed with toluene. Cysteine hydrochloride monohydrate (29.7 g) andaqueous sodium hydroxide solution were added to the above aqueous layer.The reaction mixture was stirred at room temperature till the completeconversion of7-chloro-2-[[(1S)-2,2-dimethylcyclopropane]carboxamido]-2-heptenoic acidto the product was achieved. The corresponding E isomer was isomerizedto cilastatin by heating the reaction mixture at 85-90° C. afteradjusting the pH to 0.5 with concentrated hydrochloric acid.

Purification of Cilastatin

The reaction mixture obtained above was loaded on a column packed withdiaion HP 20 resin as the adsorbent. The column was washed with water toremove sodium chloride and then the product was eluted with aqueousacetonitrile. The column fractions containing the pure product werepooled and concentrated to obtain pure cilastatin (16.3 g; Purity: 99.2%by HPLC).

While the present invention has been described in terms of its specificembodiments, certain modifications and equivalents will be apparent tothose skilled in the art and are intended to be included within thescope of the present invention.

1. A process for the purification of cilastatin which comprisescontacting a solution of crude cilastatin with a non-ionic adsorbentresin and recovering pure cilastatin from a solution thereof.
 2. Theprocess of claim 1 wherein the solution of crude cilastatin is obtaineddirectly from the reaction mixture.
 3. The process of claim 1 whereinthe solution of crude cilastatin is obtained by dissolving crudecilastatin in a suitable solvent.
 4. The process of claim 3 wherein thesuitable solvent is selected from water, an organic solvent, andmixture(s) thereof.
 5. The process of claim 4 wherein the organicsolvent is selected from the group consisting of methanol, ethanol,acetonitrile and acetone.
 6. The process of claim 1 wherein thenon-ionic adsorbent resin comprises a non-ionic macroporous waterinsoluble polymer.
 7. The process of claim 6 wherein the polymer isselected from the group consisting of polyacrylates or copolymers ofstyrene and polyvinyl benzene.
 8. The process of claim 7 wherein thepolymer is a copolymer of styrene cross linked with divinylbenzene.
 9. Aprocess for the preparation of cilastatin comprising heating a solutionof cilastatin containing the corresponding E-isomer at a pH of about 0.5to 1.5.
 10. The process of claim 9 wherein the solution is heated to85-95° C.
 11. The process of claim 9 wherein the solution is heated to85-95° C. at a pH of about 0.5.
 12. A process for the preparation ofpure cilastatin comprising: (i) heating a solution of cilastatincontaining the corresponding E-isomer at a pH of about 0.5 to 1.5, and(ii) contacting the solution obtained with a non-ionic adsorbent resinand recovering pure cilastatin from a solution thereof.